Method and kit for detection of mutations in mitochondrial dna

ABSTRACT

The present invention is within the medical field. More precisely, the invention relates to a method and kit for defection of mutations/polymorphisms in human mitochondrial DNA sequences and specifically to the use of mitochondrial DNA variants (polymorphisms) with high mutation frequency to be employed in the comparison of biological samples with samples of known origin in the purpose of, for example, human identification or forensic genetics.

FIELD OF INVENTION

The present invention is within the medical field. More precisely, the invention relates to a method and kit for detection of mutations/polymorphisms in human mitochondrial DNA sequences and specifically to the use of mitochondrial DNA variants polymorphisms) to be employed in the comparison of biological samples with samples of known origin in the purpose of, for example, human identification or forensic genetics.

BACKGROUND OF THE INVENTION

There are several methods know today for detection of mutations or polymorphisms, these can be grouped in enzymatic and non-enzymatic based methods. Non-enzymatic methods are based on hybridisation and optionally using chemical cleavage. Several patents from Affymetrix Inc., Santa Clara, Calif., USA, disclose methods where a large number of oligonucleotides are arranged on a surface, so called DNA array or DNA chips (for example, Fodor et al. U.S. Pat. No. 5,510,270). These oligonucleotide arrays are used for hybridisation of fluorescently labelled DNA and can with large number oligonucleotides, sequence and mutations can be identified. The drawback with hybridisation is that it is temperature, salt and sequence dependent and it is well known in the art that it is hard to get uniform hybridisation of many oligonucleotides at one temperature. In a situation when fluorescently labelled DNA is used as a probe the detected signal will very often differ in intensity. The generated image is then difficult to interpret and analyse.

A method called single-strand conformation polymorphism (SSCP) analysis is a intermolecular hybridisation method, where a PCR fragment is heated and quickly chilled then loaded directly on to a gel for electrophoretic separation. The drawback of this method is that they require electrophoresis, which is tedious and laboriously.

Enzymatic methods utilises an enzyme to perform the mutation detection activity, which include all sequencing methods. One method, the Enzymatic Mutation Detection technique, EMD, is a combination of hybridization and a specific heterozygote-cleaving enzyme, cleavase, this method has been developed and commercialised by Amersham Biosciences, Uppsala, Sweden. The drawback of this method is also that they require electrophoresis, which is tedious and laboriously.

Sanger sequencing or dideoxy-sequencing is the most used method for mutation discovery. A variant of this method, mini-sequencing, is used for conformation of mutations and SNP analysis. In mini-sequencing the primer is hybridised to the template just adjacent to the SNP to be studied and terminators are used in the extension reaction. The mostly used detection method is today based on dyed terminators or dyed primers, but radioactive labelled terminators or primers can also be used. Conditions and reagents for primer extension reactions are well known in the art, and are described in detail in Molecular Cloning: A laboratory manual, Sambrook et al., eds, Cold Spring Harbor Laboratory Press 1989.

Human identification has been based on analysis of either nuclear DNA or small segments of mtDNA. The studies of mtDNA, based on such materials as teeth, skeletal fragments, degraded tissue and shed hair have been focused to a small segment of the mtDNA genome, denoted the D-loop. However, in co-pending international application PCT/SE01/01691 the entire mtDNA for the purpose of human identification has been described. In this international application about 1500 polymorphic sites in the mtDNA are listed. There is no description of mutation frequency and no teachings of how to select a set of preferred polymorphic sites.

SUMMARY OF THE INVENTION

This present invention is based on sequencing or a sequencing-by-synthesis technique and a set of primers for detection of polymorphic sites in a human mitochondrial genome. A brief description of sequencing-by-synthesis; this method was first described by Melamede and if fully described in U.S. Pat. No. 4,863,849, in short an activated nucleotide with radioactivity or a dye is added together with a DNA elongating agent to a primer-template complex and allowed to elongate. After elongation, the activated nucleotide is removed and detection is done to determine if the activated nucleotide is incorporated or not, in next step another activated nucleotide is added and elongation is allowed again, these steps are repeated until the DNA sequence of interest is determined. Different variants of sequencing-by-synthesis have been proposed, such as the Pyrosequencing™ technology developed and sold by Pyrosequencing AB, Sweden, and variants with fluorescent dyes attached to the nucleotide triphosphate at different positions.

Under specified conditions, in dideoxy-sequencing set up, when high concentrations of dideoxynucleotides are used a short stretch of DNA will be sequenced. This approach can be used in combination with the sequencing-by-synthesis primers as described in Table 1 in the present invention.

One variant of sequencing-by-synthesis is presented in U.S. Pat. No. 5,302,509 by Cheeseman, where a primer is attached to a substrate, ssDNA is hybridised thereto as a template and an added dNTP having a blocking-detectable group at the 3′-end. If the blocked-detectable dNTP is incorporated in the growing chain it can be detected, if detected the blocking group will be removed and a new blocked-detectable dNTP is added, these steps are repeated and the sequence can be deduced. Similar approaches of sequencing-by-synthesis are shown in WO 93/21340 and WO 00/53812, but here the detectable group is attached at other positions and with different linkers to the dNTP molecule. In particular WO 00/53812 disclose an array format of a sequencing-by-synthesis, which can be used in combination with the present invention. The application WO 00/53812 is hereby incorporated as a reference.

Another variant of sequencing-by-synthesis is the Pyrosequencing method, which is developed at the Royal Institute of Technology in Stockholm (Ronaghi et al. 1998, Alderborn et al. 2000). The principle of the Pyrosequencing reaction: A single stranded DNA fragment (attached to a solid support), carrying an annealed sequencing primer acts as a template for the Pyrosequencing reaction. In the first two dispensations, substrate and enzyme mixes are added to the template. The enzyme mix consists of four different enzymes; DNA polymerase, ATP-sulphurylase, luciferase and apyrase. The nucleotides are sequentially added one by one according to a specified order dependent on the template and determined by the user, this order is called dispensation order. If the added nucleotide is matching the template, the DNA polymerase will incorporate it into the growing DNA strand. By this action, pyrophosphate, PPi, will be released. The ATP-sulphurylase converts the PPi into ATP, and the third enzyme, luciferase, transforms the ATP into a light signal. Following these reactions, the fourth enzyme, apyrase, will degrade the excess nucleotides and ATP molecules, and the template will at that point be ready for next reaction cycle, i.e. another nucleotide addition. No light signal will be produced unless a correct nucleotide is incorporated. The PSQ instruments have been developed by Pyrosequencing AB in order to automate the sequencing reaction and monitor the light release. The PSQ instrument software presents the results as peaks in a pyrogram™, where the height of the peaks corresponds to the number of nucleotides incorporated. Dedicated software has been developed for SNP analysis and for sequencing of shorter DNA stretches, 20 up to 40 bases even up to 200 bases have in some situation been shown.

Compared to other techniques used for detection of polymorphic sites, such as hybridisation techniques, mini-sequencing, SSCP, sequencing-by-synthesis methods present some strong advantages. One is its ability to confirm that the correct polymorphism is examined, by presenting the surrounding sequence and not only the polymorphism/s. Another advantage is the flexibility in primer design, i.e. the primer can be situated up to 50 nucleotides from the variable site(s), where in mini-sequencing the primer has to be adjacent to the polymorphic site. Furthermore, sequencing-by-synthesis methods are rapid and direct sequencing techniques, which is benefit compare to SSCP, EMD and dideoxy-sequencing, which all requires electrophoresis a relative slow and indirect detection method.

The present invention provides a method for detection of mutations/polymorphisms in human mtDNA based on analysis of biological samples. According to the present invention, the identification is based on the analysis of genetic variation in the mitochondrial DNA (mtDNA) and comparison of the sample under investigation with that of known origin or with a database. Such analyses are useful in forensic casework, missing person identification, maternity investigations and in immigration investigations as well as in medical research.

Thus, in a first aspect the present invention relates to a method detection of mutations/polymorphisms in human mtDNA by determining the biological origin of a human tissue sample comprising the following steps:

-   a) determining the sequence surrounded and including the polymorphic     sites having a frequency of mutation of at least 3-4% in the     population according to Table 1 in the nucleic acid sequence of the     mitochondrial genome in said sample from a human subject; and -   a) relating the information from step a) to mitochondrial nucleic     acid sequence information of known origin.

Alternative: determining polymorphisms in a certain position showing a frequency of at least 4% of the less common variant in the population according to Table 1 or showing a high frequency within the Caucasian subgroup.

The body sample referred to above can be derived from body fluid or tissue.

Preferably, also polymorphisms showing a high degree of variation within the Caucasian population are determined in step a).

In a preferred embodiment a fragment is selected such that at least one of the studied polymorphic sites has a frequency of at least 5%, preferably at least 10% and most preferably at least 15%.

The known information in step b) may be derived from human subjects of known identity (reference subjects). Alternatively, the known information in step b) is derived from a database of nucleic acid sequence information from humans of diverse origin.

In the method of the invention one or more of the mitochondrial fragments in Table 1 are selected for determination. The selected fragments are selected such that they should have at least one polymorphic site showing a frequency of at least 3-15% or harbouring polymorphisms of special interest with the Caucasian population. These fragments are 1, 4, 12, 14, 15, 16, 19, 20, 24, 25, 26, and fragment 27, according to Table 1. The fragment sizes indicated in Table are only a suggestion and can be changed according to the sequencing strategy chosen or variation frequency data obtained from a larger population set. Any forward or reverse primers (denoted F and R in Table 2) within each fragment can be combined to be the amplification primers for each fragment.

The polymorphic sites can alternatively be detected by a method or assays such as DNA hybridisation assays (ASO, SSO hybridisation, DNA microchip, padlock), enzymatic ligation assays (OLA, padlock) enzymatic cleavage assays (EMD, Taqman), enzymatic extension assays (mini-sequencing) or other assays for typing of genetic polymorphisms.

Preferably, the mitochondrial nucleic acid sequence is determined by sequencing-by-synthesis or alternatively with sequencing or preferably by a pyrosequencing technique.

The primers listed in Table 1 are preferably used. These primers have been optimized for use in the methods of the invention; it will be notable that some modification of some or all of these primers in Table 1 may be possible without adversely affecting their performance in the methods of the invention. Such modifications may be, one or more of the nucleotides, may be substituted for other (non-complementary) nucleotides. Furthermore, each primer may be expanded or deleted 1, 2, 3, 4 or even up to 5 nucleotides at the 3′ end or the 5′ end of a primer. Thus the primer can be up to 10 bases longer at the most. This can be done due to the nature of a sequencing-by-synthesis method.

In a second aspect, the present invention provides a kit for detecting the mutations/polymorphism in the human mtDNA, comprising means for analysis of the polymorphic sites having a frequency of mutation of at least 3-4% according to Table 1, preferably at least 5%, more preferably at least 10%, most preferably at least 15%.

The kit may comprise one or more of the primers in Table 1.

In a preferred kit that will perform the method of the invention the selected fragments are 1, 4, 12, 14, 15, 16, 19, 20, 24, 25, 26, and fragment 27, according to Table 1. The fragment sizes indicated in Table are only a suggestion and can be changed according to the sequencing strategy chosen. Any forward or reverse primers (denoted F and R in Table 2) within each fragment can be combined to be the amplification or sequencing primers for each fragment.

The means for analysis may be sequencing-by-synthesis reagents, sequencing reagents or pyrosequencing reagents.

I one embodiment two or more of the sequencing primers in Table 1 are attached to a solid support, such as a microtiterplate well or array. Such an array or microtiterplate with sequencing primers attached can be regarded as a component in a kit.

DETAILED DESCRIPTION OF THE INVENTION

A Preferred Performance of the Present Invention

One hundred and thirty three polymorphic sequence sites where selected from the PCT application PCT/SE01/01691 on the basis that the frequency of the mutations should be higher than 4% in the material of 124 completely sequenced human mitochondrial genomes, some additional mutations has also been included with lower frequency, since they are informative in different populations, more specifically in a Caucasian population. These 133 mutations are located on 27 PCR fragments. The fragments are relatively short which enables analysis of degraded sample material.

Method:

One or all of the 27 PCR fragments are amplified, with two primers, where one of the primers contains means for attachment, exemplified with the streptavidin—biotin binding par. After amplification the fragment is attached to a support, which can be a solid or porous bead, a surface, such as plastic, silica or similar surface. Two, three or several DNA fragments can be attached to one surface and the can also be arrayed.

After the bind to a support one strand is removed, by temperature or high pH, at least one primer from Table 1 is annealed.

An alternative way to perform the invention is, first binding at least two sequencing primers selected from Table 1 to a solid support, secondly hybridising at least one of the amplified fragments from Table 1.

The primer in the template/primer complex is extended in a sequencing or a sequencing-by-synthesis reaction. The sequence will be generated and thereby the polymorphism will be identified.

Kit:

A kit containing amplification primers and primers as described in Table 1, for a sequencing or a sequencing-by-synthesis reaction.

A kit containing amplification and sequence primers as described in Table 1, selected is such a way that the frequency of less common variant is higher then 10%, 5%, 4% or 2%, and sequencing-by-synthesis primers as described in Table 1 for the corresponding mutations. Optionally, reagents for sequencing or sequencing-by-synthesis can be included in the kit. TABLE 1 Polymorphic positions and frequencies No of p.m./124 Polymorphism Fragm. Fragm. Nt Change samples frequency No Size′ 316 G −> A 6 5%  1* 456 C −> T 4 3%  1* 462 C −> T 1 1%  1* 489 T −> C 30 24%   1* 514 CA ins/del 42 34%   1* 265 709 G −> A 11 9% 2 769 G −> A 15 12%  2 825 T −> A 12 10%  2 1018 G −> A 15 12%  2 1048 C −> T 7 6% 2 399 1719 G −> A 7 6% 3 1888 G −> A 4 3% 3 223 2706 A −> G 114 92%   4* 2758 G −> A 12 10%   4* 2885 T −> C 12 10%   4* 3010 G −> A 13 10%   4* 3027 T −> C 3 2%  4* 373 3516 C −> A 4 3% 5 3552 T −> A 5 4% 5 3594 C −> T 15 12%  5 3666 G −> A 7 6% 5 3796 A −> T 5 4% 5 331 4104 A −> G 15 12%  6 4117 T −> C 9 7% 6 4216 T −> C 5 4% 6 4312 C −> T 5 4% 6 302 4586 T −> C 5 4% 7 4715 A −> G 5 4% 7 4917 A −> G 4 3% 7 391 5263 C −> T 4 3% 8 5442 T −> C 5 4% 8 5460 G −> A 15 12%  8 5465 T −> C 11 9% 8 252 7028 C −> T 113 91%  9 7055 A −> G 7 6% 9 7146 A −> G 11 9% 9 7196 C −> A 5 4% 9 7256 C −> T 15 12%  9 7274 C −> T 2 2% 9 310 7389 T −> C 7 6% 10  7521 G −> A 16 13%  10  201 8027 G −> A 7 6% 11  8087 T −> C 4 3% 11  8251 G −> A 6 5% 11  8277 Ins/Del 19 15%  11  304 8404 T −> C 8 6% 12* 8414 C −> T 4 3% 12* 8468 C −> T 12 10%  12* 8584 G −> A 3 2% 12* 8655 C −> T 12 10%  12* 8697 G −> A 4 3% 12* 8701 A −> G 51 41%  12* 370 8790 G −> A 9 7% 13  8964 C −> T 7 6% 13  9042 C −> T 5 4% 13  9072 A −> G 6 5% 13  9103 T −> C 4 3% 13  9123 G −> A 11 9% 13  421 9347 A −> G 5 4% 14* 9540 T −> C 51 41%  14* 9545 A −> G 5 4% 14* 271 10238 T −> C 13 10%  15* 10310 G −> A 4 3% 15* 10321 T −> C 6 5% 15* 10398 A −> G 55 44%  15* 10400 C −> T 29 23%  15* 10463 T −> C 5 4% 15* 10586 G −> A 6 5% 15* 10589 G −> A 5 4% 15* 420 10664 C −> T 5 4% 16* 10688 G −> A 13 10%  16* 10810 T −> C 13 10%  16* 10819 A −> G 4 3% 16* 10873 T −> C 50 40%  16* 10915 T −> C 8 6% 16* 305 11251 A −> G 5 4% 17  11467 A −> G 5 4% 17  258 11719 G −> A 111 90%  18  11899 T −> C 6 5% 18  11914 G −> A 16 13%  18  12007 G −> A 10 8% 18  368 12239 C −> T 10 8% 19* 12308 A −> G 4 3% 19* 12372 G −> A 5 4% 19* 184 12705 C −> T 63 51%  20* 12810 A −> G 6 5% 20* 12940 G −> A 9 7% 20* 316 13105 A −> G 14 11%  21  13263 A −> G 5 4% 21  13276 A −> G 5 4% 21  13368 G −> A 5 4% 21  343 13485 A −> G 6 5% 22  13500 T −> C 10 8% 22  13506 C −> T 12 10%  22  13590 G −> A 6 5% 22  13650 C −> T 15 12%  22  13708 G −> A 5 4% 22  13789 T −> C 7 6% 22  349 13928 G −> C 8 6% 23  14000 T −> A 6 5% 23  14022 A −> G 10 8% 23  14025 T −> C 7 6% 23  14088 T −> C 7 6% 23  14148 A −> G 5 4% 23  14178 T −> C 7 6% 23  14182 T −> C 5 4% 23  338 14766 C −> T 13 10%  24* 14783 T −> C 29 23%  24* 14798 T −> C 1 1% 24* 14905 G −> A 6 5% 24* 14911 C −> T 6 5% 24* 15043 G −> A 31 25%  24* 330 15301 G −> A 39 31%  25* 15431 C −> A 5 4% 25* 15452 C −> A 5 4% 25* 15487 A −> T 5 4% 25* 259 15607 A −> G 21 17%  26* 15663 T −> C 4 3% 26* 15670 T −> C 4 3% 26* 15746 A −> G 10 8% 26* 15784 T −> C 4 3% 26* 15924 A −> G 7 6% 26* 15928 G −> A 4 3% 26* 391 16325 T −> C 4 3% 27* 16327 C −> T 6 5% 27* 16343 A −> G 6 5% 27* 16356 T −> C 4 3% 27* 16357 T −> C 9 7% 27* 16360 C −> T 8 6% 27* 16362 T −> C 19 15%  27* 16390 G −> A 8 6% 27* 16399 A −> G 5 4% 27* 16519 T −> C 69 56%  27* 259 ′Fragment size including PCR primers. *Denotes fragments with at least one polymorphism showing a frequency of at least 15% or harboring polymorphisms of special interest within the Caucasian population. Numbering of mtDNA positions is according to Anderson et al. 1981.

TABLE 2 PCR and sequencing primers. Numbers are according to Anderson et al. Primers are named by 5′ nucleotide. Frag- Tm ment Primer (° C.) Sequence (5′-3′) Nucleotides detected 1 283 F 51,3 AACAAAAAATTTCCACCAAA 316 1 351 R 48,6 TTGGCAGAGATGTGTTTAA 316 1 431 F 53,6 CACCCCCCAACTAACACA 456 462 489 514 1 465 F 51,1 CTCCCATACTACTAATCTCATC 489 514 AA 1 502 R 52,0 GGGCGGGGGTTGT 489 462 456 1 547 R 53,1 TTCGGGGTATGGGGTTA 514 489 462 456 2 676 F 52,3 GCTCTTAGTAAGATTACACATGCA 709 769 2 740 R 54,7 CGTGGTGATTTAGAGGGTGA 709 2 741 F 55,9 ATCAAAAGGGACAAGCATCAA 769 825 2 790 R 52,8 TAAGCGTTTTGAGCTGCA 769 709 2 800 F 55,3 CACCCCCACGGGAAA 825 2 853 R 49,8 GTTAAACTTTCGTTTATTGCTAA 825 769 2 994 F 51,2 AAAAACTCCAGTTGACACAAA 1018 1048 2 1074 R 53,3 CCCAGTTTGGGTCTTAGCTA 1048 1018 3 1691 F-a 53,2 CACTCCACCTTACTACCAGACA 1719 3 1691 F-b 54,8 CACTCCACCTTACTACCAGACAA 1719 3 1752 R 53,1 ATCGCCTATACTTTATTTGGGTA 1719 3 1856 F 53,5 ATGAATTAACTAGAAATAACTTTG 1888 CAA 3 1913 R 54,6 CTGGTTTCGGGGGTCTTA 1888 4 2680 F 51,3 TGACCTGCCCGTGAA 2706 2758 4 2724 F 51,3 GACCCTATGGAGCTTTAATTTA 2758 4 2733 R 52,2 CCATAGGGTCTTCTCGTCTT 2706 4 2782 R 51,8 TAGGACCTGTGGGTTTGTTA 2758 2706 4 2861 F 52,9 ACTTCACCAGTCAAAGCGA 2885 4 2913 R 50,1 TGGTCAAGTTATTGGATCAA 2885 4 2987 F 54,2 TCGATGTTGGATCAGGACA 3010 3027 4 3052 R 50,7 TTAATCGTTGAACAAACGAA 3027 3010 5 3493 F 54,8 CCGCCACATCTACCATCA 3516 3552 3594 5 3574 R 54,2 GGAGGGGGGTTCATAGTAGA 3552 3516 5 3569 F 53,2 CCCTCCCCATACCCAA 3594 3666 5 3631 F 53,5 TCTAGCCTAGCCGTTTACTCA 3666 5 3637 R 53,6 GGCTAGAGGTGGCTAGAATAAA 3594 3552 3516 5 3691 R 52,5 GGGCGTAGTTTGAGTTTGA 3666 3594 5 3764 F 52,5 CATTACTAATAAGTGGCTCCTTT 3796 AA 5 3823 R 52,8 AGAGGTGTTCTTGTGTTGTGATA 3796 6 4054 F 54,8 CTCTCCCCTGAACTCTACACAA 4101 4117 6 4141 R 55,5 GGGGGTATGCTGTTCGAA 4117 4101 6 4185 F 52,3 CCTACCACTCACCCTAGCA 4216 6 4251 R 53,9 GGGAATGCTGGAGATTGTAA 4216 6 4275 F 50,2 GATAAAAGAGTTACTTTGATAGAG 4312 TAAA 6 4355 R 53,6 GGATGGGTTCGATTCTCATA 4312 7 4561 F 52,1 TAGGCGTAGAAATAAACATGCTA 4586 7 4620 R 54,1 GAGGGTTTATTTTTTTGGTTAGAA 4586 7 4676 F 50,8 CCTTCTAATAGCTATCCTCTTCA 4715 7 4741 R 52,9 TTGGTAGTATTGGTTATGGTTCA 4715 7 4880 F 55,1 CCCCATCTCAATCATATACCAA 4917 7 4951 R 52,5 GATAAGATTGAGAGAGTGAGGAGA 4917 8 5243 F 51,3 CGGCTTTTTGCCCA 5263 8 5285 R 47,4 TTTTGTGAATTGTTCGATAA 5263 8 5404 F 49,6 AAATAAAATGACAGTTTGAACATA 5442 5460 6565 8 5494 R 51,9 AAAGGGGAGATAGGTAGGAGTA 5465 5460 5442 9 6990 F 49,4 CTAGACATCGTACTACACGACA 7028 7055 9 7080 R 52,7 AGCCTCCTATGATGGCAA 7055 7028 9 7115 F 57,9 CCTAGACCAAACCTACGCCAA 7146 7196 9 7163 F 56,9 CGTAAATCTAACTTTCTTCCCAC 7196 7256 7274 AA 9 7176 R 53,1 AAGTTAGATTTACGCCGATGA 7146 7155 9 7216 R 57,3 CGGGGCATTCCGGATA 7196 7146 9 7235 F 54,9 CGATGCATACACCACATGAA 7256 7274 9 7299 R 48,8 TTACTGCTGTTAGAGAAATGAA 7274 7256 7196 10 7349 F 52,6 CCTAATAGTAGAAGAACCCTCCA 7389 10 7409 R 54,2 GTAGGGTGGGGGGCA 7389 10 7497 F 56,3 GGCCTCCATGACTTTTTCAA 7521 10 7549 R 52,5 ACAAAGTTATGAAATGGTTTTTC 7521 TA 11 8006 F 53,4 CGAGTAGTACTCCCGATTGAA 8027 8087 11 8029 F 55,2 CCCCATTCGTATAATAATTACAT 8087 CA 11 8063 R 50,5 AGACGTCTTGTGATGTAATTATTA 8027 TA 11 8113 R 53,3 GGGAATGGCATCTGTTTTTAA 8087 8027 11 8206 F 53,2 GCCCATCGTCCTAGAATTAA 8251 8277 11 8223 F 54,5 TAATTCCCCTAAAAATCTTTGAAA 8251 8277 11 8309 R 52,2 GTTAGCTTTACAGTGGGCTCTA 8277 8251 12 8359 F 56,4 CAGTGAAATGCCCCAACTAAA 8404 8414 8468 12 8435 F 53,8 ACCCAACTAAAAATATTAAACACA 8468 AA 12 8438 R 50,4 GGGTGATGAGGAATAGTGTAA 8414 8404 12 8488 R 53,6 GGGCTTTGGTGAGGGA 8468 8414 8404 12 8549 F 55,5 CATTCATTGCCCCCACA 8584 8655 12 8621 R 56,5 GGGATCAATAGAGGGGGAAA 8584 12 8632 F 51,4 TATCTCATCAACAACCGACTAA 8655 8697 9701 12 8696 R 54,4 ATTTGTTTTGAGGTTAGTTTGATT 8655 8584 AGT 12 8728 R 55,8 AGGTTCGTCCTTTAGTGTTGTGTA 8701 8697 8655 13 8748 F 52,9 CTTAATCATTTTTATTGCCACAA 8790 13 8822 R 55,2 GATAGTTGGGTGGTTGGTGTAA 8790 8701 13 8932 F 55,1 CCCCTTATCCCCATACTAGTTATT 8964 9042 ATTA 13 8995 R 53,1 CCAGGGCTATTGGTTGAA 8964 13 9052 F 54,8 AGCGCCACCCTAGCAA 9072 9103 9123 13 9091 F 50,9 ACACTTATCATCTTCACAATTCT 9123 AA 13 9107 R 54,7 GTGAAGATGATAAGTGTAGAGGG 9072 9042 AA 13 9168 R 52,8 GAAAACGTAGGCTTGGATTAA 9123 9103 9072 14 9305 F 54,6 GTGATTTCACTTCCACTCCATAA 8347 14 9382 R 56,2 CGCCATCATTGGTATATGGTTA 8347 14 9525 F 56,0 GCCCCTACCCCCCAA 9540 9545 14 9575 R 54,5 CGGGGTGATGCCTGTT 9545 9540 15 10205 F 55,4 CCCTTTCTCCATAAAATTCTTCT 10238 10310 10321 TA 15 10272 R 54,7 GGAGGGCAATTTCTAGATCAA 10238 15 10281 F 55,6 CTACCATGAGCCCTACAAACAA 10310 10321 10398 10400 15 10357 R 50,3 AGGGCTAGGATGATGATTAA 10321 10310 10238 15 10362 F 55,0 CTGGCCTATGAGTGACTACAAAA 10398 10400 10463 15 10426 F 53,5 CGAATGATTTCGACTCATTAAA 10463 15 10436 R 50,6 GAAATCATTCGTTTTGTTTAAA 10400 10398 15 10518 R 53,6 GAAGTGAGATGGTAAATGCTAGTA 10463 10400 10398 TAA 15 10540 F 53,8 CACACCTCATATCCTCCCTACTA 10586 10589 15 10624 R 56,6 TGGGTGTTGAGGGTTATGAGA 10589 10586 16 10636 F 57,2 CCAATATTGTGCCTATTGCCA 10664 10688 16 10644 F 45,3 GTGCCTATTGCCATACTA 10664 10688 16 10715 R 52,7 GGAGATGAGACTAGTAGGGCTA 10688 10664 16 10776 F 50,0 TCCCAACAATTATATTACTACCA 10810 10819 10873 16 10845 R 51,6 GTGGTTGTGTTGATTCAAATTA 10819 10810 16 10847 F 51,5 CACAGCCTAATTATTAGCATCA 10873 10915 16 10905 R 51,1 AGGTTGTTGTTGATTTGGTTA 10873 10819 10810 16 10938 R 45,1 GGGTCGGAGGAAAA 10915 10873 16 10940 R 55,2 GGGGGTCGGAGGAAAA 10915 10873 17 11227 F 49,0 CTCCCTTCCCCTACTCA 11251 17 11274 R 53,2 CCTAGGGTGTTGTGAGTGTAAA 11251 17 11430 F 50,7 CCATCGCTGGGTCAA 11467 17 11484 R 49,9 CCATAGCCGCCTAGTTT 11467 18 11689 F 58,5 CGGCGCAGTCATTCTCATAA 11719 18 11690 F 53,4 GGCGCAGTCATTCTCATAA 11719 18 11744 R 53,4 GGCAGAATAGTAATGAGGATGTAA 11719 18 11861 F 54,5 GCCTTACCCCCCACTATTAA 11899 11914 18 11946 R 52,0 GTAAGTAGGAGAGTGATATTTGAT 11914 11899 CA 18 11980 F 54,1 CCTCTACATATTTACCACAACAC 12007 AA 18 12053 R 57,3 GTGTGAATGAGGGTTTTATGTTGT 12007 TA 18 12056 R 54,1 CTCGTGTGAATGAGGGTTTTA 12007 19 12208 F 54,3 AGAAAGCTCACAAGAACTGCTAA 12239 12308 19 12250 F 56,5 CAACATGGCTTTCTCAACTTTTAA 12308 12372 19 12268 R 53,1 AGTTGAGAAAGCCATGTTGTTA 12239 19 12345 F 51,9 GCACACTACTATAACCACCCTAA 12372 19 12364 R 51,5 GGGTGGTTATAGTAGTGTGCA 12308 19 12391 R 54,7 TGGGGGGAATTAGGGAA 12372 12308 20 12651 F 51,1 GTGATATATAAACTCAGACCCAAA 12705 20 12737 R 52,4 GCGGTAACTAAGATTAGTATGGT 12705 AA 20 12764 F 55,1 GCTGAGAGGGCGTAGGAA 12810 20 12862 R 50,9 GGTTGTATAGGATTGCTTGAA 12810 20 12918 F 53,0 CTCATGAGACCCACAACAAA 12940 20 12966 R 51,6 AGGCTTGGATTAGCGTTTA 12940 21 13066 F 52,8 TCAGCCCTACTCCACTCAA 13105 21 13126 R 51,6 GGAAGCGGATGAGTAAGAA 13105 21 13239 F 54,3 CGTAGCCTTCTCCACTTCAA 13263 13276 21 13298 R 52,7 TGGTTGATGCCGATTGTA 13276 13263 21 13328 F 54,1 CCCACGCCTTCTTCAAA 13368 21 13408 R 52,5 TTCGAATATCTTGTTCATTGTTA 13368 22 13465 F 51,7 AGCCTAGCATTAGCAGGAA 13485 13500 13506 22 13525 R 53,8 CGATGATGTGGTCTTTGGA 13506 13500 13485 22 13551 F 53,6 CGCCTGAGCCCTATCTATTA 13590 13650 22 13596 F 52,3 CGCCTATAGCACTCGAATAA 13650 22 13636 R 52,2 GACCTGTAGGGTGAGAAGAA 13590 22 13680 R 51,9 GGGGTTATTTTCGTTAATGTTA 13650 13708 22 13680 F 53,9 CACCCTACTAAACCCCATTAAA 13708 13789 22 13757 R 52,3 GGGGAAATGTTGTTAGTAATGA 13708 13650 22 13763 F 55,4 CCCCCTTCCAAACAACAA 13789 22 13810 R 50,8 CGAGGGCTGTGAGTTTTA 13789 13708 22 13813 R 51,2 CAGCGAGGGCTGTGA 13789 13708 23 13880 F 50,6 CCCCACTATGCACATTTTA 13928 23 13902 F 50,8 CTCCAACATACTCGGATTCTA 13928 14000 14022 23 13972 R 55,1 GGCTCGTAAGAAGGCCTAGA 13928 23 13977 F 55,5 CCTGCCCCTACTCCTCCTA 14000 14022 14025 14088 23 14053 R 54,5 TGGAGATTTGGTGCTGTGA 14025 14022 14000 23 14061 F 53,0 CATCACCTCAACCCAAAAA 14088 14148 14178 23 14116 R 53,5 GGAAGAAGAAAGAGAGGAAGTAAA 14088 14025 14022 14000 23 14119 F 51,0 CTCATCCTAACCCTACTCCTAA 14148 14178 14182 23 14217 R 50,4 GTAGTAGTTACTGGTTGAACATT 14182 14178 14148 GT 24 14748 F 52,7 TGACCCCAATACGCAAA 14766 14783 14798 24 14835 R 53,0 CATGCGGAGATGTTGGA 14798 14783 14766 24 14862 F 55,6 CCTGCCTGATCCTCCAAA 14905 14911 24 14950 R 53,9 GTGGGCGATTGATGAAAA 14911 14905 24 15001 F 54,8 TGGCGCCTCAATATTCTTTA 15043 24 15077 R 52,0 CTGAGTAGAGAAATGATCCGTAA 10543 25 15271 F 51,8 CACACGATTCTTTACCTTTCA 15301 25 15328 R 55,8 TGTTGCTAGGGCTGCAATAA 15301 25 15402 F 53,1 CCTTCCACCCTTACTACACAA 15431 15452 15487 25 15454 F 51,1 TCTCTCCTTAATGACATTAACAC 15487 TA 25 15493 R 53,3 GAGGTCTGGTGAGAATAGTGTTAA 15452 15431 25 15529 R 52,9 GGGGTTGGCTAGGGTATAA 15487 15452 15431 26 15588 F 51,3 TCCGATCCGTCCCTAA 15607 15663 15670 26 15636 F 53,2 CCATCCTCATCCTAGCAATAA 15663 15670 15746 26 15652 R 51,8 TGCTAGGATGAGGATGGATA 15607 26 15710 R 51,5 GGCTTAGTGGGCGAAA 15670 15663 15607 26 15722 F 52,1 TGACTCCTAGCCGCAGA 15746 26 15758 F 51,5 ATCGGAGGACAACCAGTAA 15784 26 15770 R 55,4 GTTGTCCTCCGATTCAGGTTA 15746 15670 15663 26 15807 R 53,3 GCTACTTGTCCAATGATGGTAA 15784 15746 26 15882 F 52,4 GGGCCTGTCCTTGTAGTATAA 15924 15928 26 15978 R 50,6 GGAGTTAAAGACTTTTTCTCTGA 15928 15924 27 16291 F 54,1 CCACCCTTAACAGTACATAGTACA 16325 16327 16343 16356 16357 16360 16362 16390 16399 TAA 27 16369 F 55,8 GGATGACCCCCCTCAGATA 16390 16399 27 16384 R 56,1 CTGAGGGGGGTCATCCA 16362 16360 16357 16356 16343 16327 16325 27 16439 R 54,2 GCACTCTTGTGCGGGATA 16399 16390 16362 16360 16357 16356 16343 16327 16325 27 16495 F 54,0 CGACATCTGGTTCCTACTTCA 16519 27 16549 R 56,3 GGGGAACGTGTGGGCTA 16519 Nucleotides detected denotes specific polymorphisms detected within 50 basepairs from the primer.

Polymorphisms shown italic denotes polymorphisms that can be detected within 100 basepairs from the primer (for fragments where longer readlengths can be obtained or when a manually programmed dispension order is used. 

1. A method for detection of mutations/polymorphisms in a sample of human mitochondrial DNA comprising the following steps: (a) determining the presence or absence of polymorphic sites having a frequency of mutation of less than 3% in the general population but at least 3% in the Caucasian population according to Table 1 in the nucleic acid sequence of the mitochondrial genome in said sample from a human subject; and (b) relating the information from step (a) to mitochondrial nucleic acid sequence information of known origin; and (c) relating the information from step (a), where in one or more of the mitochondrial fragments 1, 4, 12, 14, 15, 16, 19, 20, 24, 25, 26, and 27 in Table 1, to be used for determination of polymorphic site(s).
 2. A method according to claim 1, wherein the frequency of mutations is at least 5%.
 3. A method according to claim 1, wherein the known information in step (b) is derived from a database of nucleic acid sequence information from humans of diverse origin.
 4. A method according to claim 1, wherein the polymorphic sites are detected by assays such as DNA hybridization assays (ASO, SSO hybridization, DNA microchip, padlock), enzymatic ligation assays (OLA, padlock), enzymatic cleavage assays (EMD, Taqman), enzymatic extension assays (mini-sequencing) or other assays for typing of genetic polymorphisms.
 5. A method according to claim 1, wherein the mitochondrial polymorphic sites(s) is/are determined by sequencing.
 6. A method according to claim 5, wherein the sequencing method is sequencing-by-synthesis.
 7. A method according to claim 5, wherein the sequencing method is pyrosequencing.
 8. A method according to claim 1, using the primers listed in Table
 2. 9. A kit for detecting the detecting mutations/polymorphism in the human mtDNA, comprising means for analysis of the polymorphic sites having a frequency of mutation of at least 3% according to Table
 1. 10. A method according to claim 9, comprising means for analysis of the polymorphic sites having a frequency of mutation of at least 5% according to Table
 1. 11. A kit according to claim 9, comprising one or more of the sequencing primers in Table
 2. 12. A kit according to claim 9, comprising two or more amplification primers according to Table 2 for fragment 1, 4, 12, 14, 15, 16, 19, 20, 24, 25, 26, and 27 according to Table
 1. 13. A kit according to claim 9, wherein the means for analysis are sequencing-by-synthesis reagents.
 14. A kit according to claim 9, wherein the means for analysis are sequencing reagents.
 15. A kit according to claim 9, wherein the means for analysis are pyrosequencing reagents.
 16. A kit according to claim 11, wherein two or more of the sequencing primers in Table 2 are attached to a solid support, such as a microtierplate well or array.
 17. A method according to claim 1, wherein the frequency of mutations is at least 10%.
 18. A method according to claim 1, wherein the frequency of mutations is at least 15%.
 19. A kit according to claim 9, comprising means for analysis of the polymorphic sites having a frequency of mutation of at least 10% according to Table
 1. 20. A kit according to claim 9, comprising means for analysis of the polymorphic sites having a frequency of mutation of at least 15% according to Table
 1. 